Sab5500134

Tissue slides were stained as previously described on the paraffin‐embedded slides (Damhofer et al., 2015). Antibodies and dilutions used were CK19 1 : 500 (MU246‐UC; Biogenex), CXCR4 1 : 400 (Ab124824; Abcam, Cambridge, UK), Ki67 1 : 2000 (SAB5500134; Sigma). For picrosirius red staining, slides were deparaffinized, stained in a 0.1% picrosirius red solution (Sigma) in saturated picric acid for 1 h, and washed three times with 0.1 m acetic acid solution. All slides were imaged on an Olympus BX51 (Tokyo, Japan). Quantification of Ki67 staining was performed with Fiji count particles (Schindelin et al., 2012), after DAB/H color deconvolution. For IF images, slides were cut at 10 μm, mounted in Prolong Gold (ThermoFisher), and imaged on an EVOS fluorescence microscope (ThermoFisher). For collagen staining in tissue culture vessels, cells were cultured as described and after 7 days of coculture washed with PBS three times prior to fixation in 4% paraformaldehyde for 15 min. Following three washes with PBS, cells were stained for their collagen deposition as described above for 18 h and were equally treated as the tissue slides. Cells were subsequently imaged on an Olympus BX51. Quantifications of the percentage of Ki67‐positive nuclei, width of HE staining, or percentage of Venus‐positive cells were performed using Fiji package of imagej.

Spheroids were taken off-chip for histopathology by removing the top part of the chip, by inserting tweezers between the top part and the adhesive tape, without touching the channel with the tweezers. A PDMS ring with an inner diameter of 8 mm and a thickness of 3 mm was placed around the well and gently filled with 150 µL of PBS. The spheroid was harvested with the p200 wide bore pipette tip and transferred to 4% paraformaldehyde (PFA, Sigma, USA) in PBS. Spheroids were placed in 70% alcohol overnight, dehydrated, transferred to a biopsy foam pad, embedded in paraplast, and cut into 0.4 µm sections. Tissue sections were rehydrated and stained with hematoxylin and eosin (H&E) to evaluate the tissue structure or a diaminobenzidine (DAB) staining for apoptosis (cleaved Caspase-3) or proliferation (Ki67) with a hematoxylin counterstain. Antibodies used for the DAB staining were primary antibodies cleaved Caspase-3 (ASP-175) (1:1000, Cell Signaling #9661, USA) or Ki67 (1:1000, Sigma #SAB5500134, USA) and secondary anti-rabbit antibody linked to horseradish peroxidase (HRP). Slides were dehydrated and mounted with Pertex (VWR, Amsterdam, The Netherlands).

FFPE samples were used for Ki67 staining. Sections of a thickness of 4 µm were prepared and deparaffinised using xylene and rehydrated through ethanol. Antigen retrieval was achieved using 10 mM sodium citrate buffer (pH = 6) (Vector Laboratories, Burlingame, CA, USA) for 20 min at 98 °C. Samples were blocked using Dako REAL Peroxidase-Blocking Solution (Agilent technologies, Santa Clara, CA, USA) for 5 min at room temperature. Ki67 antibody (Sigma, SAB5500134, Saint Louis, MI, USA) was diluted 1:1000, in normal antibody diluent (Klinipath, ABB999, Duiven, The Netherlands), and incubated overnight at 4 °C. After washing with PBS, poly HRP-anti Rabbit IgG (Bright vision, DPVR-55HRP, Immunologic, Duiven, The Netherlands) was added for 30 min at room temperature and finally stained using Bright DAB solution (3,3′ diaminobenzidine, Immunologic, Duiven, The Netherlands). Counterstaining with haemotoxylin (Klinipath, 4085–9002, Duiven, The Netherlands) was incubated for 1 min. After dehydration, slides were mounted using Pertex (HistoLab, Västra Frölunda, Sweden). For material from one patient (patient 9), no staining could be performed due to low quality of the material. For quantification of stainings, haemotoxylin colour was separated from DAB using the plugin “color deconvolution” in ImageJ. Positive nuclei were calculated as a percentage of total nuclei.